Inhibition of profibrotic microRNA-21 affects platelets and their releasate
Temo Barwari, … , Yajaira Suárez, Manuel Mayr
Temo Barwari, … , Yajaira Suárez, Manuel Mayr
Published November 2, 2018
Citation Information: JCI Insight. 2018;3(21):e123335. https://doi.org/10.1172/jci.insight.123335.
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Research Article Cardiology Cell biology

Inhibition of profibrotic microRNA-21 affects platelets and their releasate

Abstract

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21–null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21–null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.

Authors

Temo Barwari, Seda Eminaga, Ursula Mayr, Ruifang Lu, Paul C. Armstrong, Melissa V. Chan, Mahnaz Sahraei, Marta Fernández-Fuertes, Thomas Moreau, Javier Barallobre-Barreiro, Marc Lynch, Xiaoke Yin, Christian Schulte, Ferheen Baig, Raimund Pechlaner, Sarah R. Langley, Anna Zampetaki, Peter Santer, Martin Weger, Roberto Plasenzotti, Markus Schosserer, Johannes Grillari, Stefan Kiechl, Johann Willeit, Ajay M. Shah, Cedric Ghevaert, Timothy D. Warner, Carlos Fernández-Hernando, Yajaira Suárez, Manuel Mayr

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Figure 1

Transfections of cardiac fibroblasts with miR-21 mimic and inhibitor.

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Transfections of cardiac fibroblasts with miR-21 mimic and inhibitor. (A...
(A) Cardiac fibroblasts (CFs) were isolated from wild-type mice and transfected with miR-21 mimic or LNA-21 (inhibitor), followed by stimulation with TGF-β1 or control treatment. Overexpression and inhibition were confirmed by qPCR (n = 8 for each transfection condition; Wilcoxon matched-pairs signed-rank test; lines and error bars represent median [IQR]; note that in 3 samples miR-21 was undetectable after transfection with LNA-21). (B) Immunoblotting for several extracellular matrix (ECM) proteins showed effects of TGF-β1 treatment but not of miR-21 mimic or inhibitor transfection (n = 4 for each condition). Ponceau S staining was used as loading control. C, control mimic/LNA; 21, miR-21 mimic/LNA-21; Mr, relative mass. TGF-β1 +/– indicates treatment 48 hours prior to conditioned media collection. (C) Proteomic analysis of the CF secretome after transfections with miR-21 mimic or inhibitor identified no significant changes in the 20 most abundant ECM proteins. Four biological replicates were analyzed for each transfection type in the presence or absence of TGF-β1 treatment. No statistically significant difference was seen between miR-21 mimic or inhibitor and its respective control for any of the shown proteins, using a FDR < 0.05, calculated with the Empirical Bayes method.